4022 2021 2021 eng 105918 12 October 186 article 0 2021-12-14 -- -- Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of L-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency. Protein Expression and Purification 10.1016/j.pep.2021.105918 1046-5928 urn:nbn:de:hbz:1010-opus4-40229 false true © Alle Rechte vorbehalten Tobias Heinks Anette Hettwer Christian Hiepen Christoph Weise Marcel Gorka Petra Knaus Thomas D. Müller Angelika Loidl-Stahlhofen eng uncontrolled Bone morphogenetic protein 2 eng uncontrolled Aggregation-prone eng uncontrolled In-silico-design eng uncontrolled Hydrophilicity enhanced hBMP2 variant eng uncontrolled E. coli SHuffle® T7 eng uncontrolled pH-shift elution Ingenieur- und Naturwissenschaften Westfälische Hochschule Gelsenkirchen Bocholt Recklinghausen https://opus4.kobv.de/opus4-w-hs/files/4022/Optimized expression and purification of a soluble BMP2 variant based on in silico design.pdf